Hematopoiesis is a critical process in both the development of mammalian embryos and sustaining of life in young and adult animals. Faulty blood-forming capabilities during early-to mid-development of an embryo can lead to pre-natal death, while deficiencies or uncontrolled blood cell differentiation and proliferation in postnatal life can lead to a wide range of dysfunction and disease including various anemias, blood leukemias/lymphomas, and immune deficiencies secondary to aberrant development of the B- and T- cell lineages. There currently exist two theories regarding the progenitor cell that leads to formation of early hematopoietic cells;the hemangioblast (a bi-potent progenitor cell that gives rise to both hematopoietic stem cells (HSC) and the hemogenic endothelium (a primitive endothelial cell that resides in the developing vasculature and differentiates into blood-forming cells within the vessel lumen). Previous work in our lab has defined a population of cKit+ cells that reside within the vascular endothelium within the developing yolk sac, and when sorted by cKit+ CD45- cell surface marker expression give rise to hematopoietic colonies when cultured in MethoCult M3434 media. While the work conducted in our lab has focused primarily on the yolk sac due to its simplicity of isolation and relative paucity of cell types, we are now eager to test our findings in the embryo proper, where sites of definitive hematopoiesis reside. The goal of the research proposed is to test the hypothesis that Hoechst dye-effluxing (SP) cells that are cKit+ Flk+ within the para-aortic splanchnopleure/aorta-gonad-mesonephros (PAS/AGM) region are hemogenic endothelial cells capable of giving rise to all blood lineages and have repopulation capabilities when transplanted into irradiated animal recipients. This hypothesis challenges the existing paradigm of the so-called hemangioblast as the predominant source of blood forming cells in the developing mammalian embryo. In order to test this hypothesis, our first aim is to isolate the SP cells from the PAS/AGM region, where definitive hematopoiesis is thought to occur, and determine their definitive hematopoietic potential in vitro. Next, we would like to use both whole mount/frozen section staining as well as in vivo imaging to track these cells throughout development and definitively show their differentiation from cells residing in the endothelium to blood-forming cells within the vessel lumen. Finally, we intend to characterize the cellular phenotype and molecular regulation of these cells and determine important/unique gene expression profiles by qRT-PCR and microarray analysis. The application of this research for public health lies in the fact that understanding the development of blood vessels and cells will aid in therapeutic approaches for human disease.